Maintenance and propagation of mesenchymal stem cells

ABSTRACT

Various embodiments of the present invention include compositions, materials and methods for maintaining and propagating mammalian mesenchymal stem cells in an undifferentiated state in the absence of feeder cells and applications of the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/297,966 filed Nov. 16, 2011, which is a divisional of U.S. patentapplication Ser. No. 11/625,763 filed Jan. 22, 2007, (now U.S. Pat. No.8,084,023). The entire contents of each of the above-referenceddisclosures are specifically incorporated herein by reference withoutdisclaimer.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was supported by the Department of Veterans Affairs, andgrants R21 AG025466 and P01 AG13938 from the National Institutes ofHealth. The government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Mesenchymal stem cells (MSCs) are characterized by their ability toproduce daughter stem cells and also to differentiate into many distinctcell types including, but not limited to, osteoblasts, stromal cellsthat support hematopoiesis and osteoclastogenesis, chondrocytes,myocytes, adipocytes of the bone marrow, neuronal cells and B-pancreaticislet cells.⁽¹⁻³⁾ Thus, MSCs are able to provide the appropriate numberof osteoblasts and stromal cells needed for bone development, boneremodeling and hematopoiesis throughout life.

MSCs are extremely rare in the bone marrow and earlier attempts toexpand them ex vivo from rodent or human marrow have proven difficult.Moreover, MSCs tend to lose their stem cell properties under traditionalcell culture conditions. This situation has impaired the use of MSCs forpractical purposes, such as, for example, therapeutic purposes.

The loss of MSC properties in vitro suggests that a critical feature ofthe marrow environment in vivo, which is responsible for the retentionof stem cell properties, is missing in standard culture systems. Thepresent invention illustrates that mesenchymal stem cells require aspecialized microenvironment or niche that supports their self-renewalcapability, and maintains their multipotentiality while facilitatingdifferentiation in response to appropriate signals.

MSCs reside within the bone marrow, which consists of stromal cells,adipocytes, vascular elements, and sympathetic nerve cells arrayedwithin a complex extracellular matirx (ECM).^((4,5)) The bone marrow ECMmay comprise molecules selected from the group consisting of collagensI, III, IV, V and VI, fibronectin, and laminin. The bone marrow ECM mayalso comprise molecules selected from the group consisting of adhesiveproteins, large molecular weight proteoglycans like syndecan andperlecan, and members of the small leucine-rich proteoglycan familyincluding biglycan and decorin.^((6,7))

The present inventors demonstrate that culture of marrow-derived MSCs ona cell-free ECM made by marrow-derived stromal cells promotesself-renewal of MSCs and helps maintain the MSCs in an undifferentiatedstate. The present inventors further demonstrate that followingexpansion on this ECM, functional MSCs were increased as evidenced byincreased formation of bone and hematopoietic marrow tissue followingsubcutaneous transplantation of in vitro expanded MSCs toimmuno-compromised mice.

Stem cells may divide asymmetrically to give a daughter stem cell and amore differentiated progeny, or symmetrically to give two identicaldaughter stem cells or two more differentiated cells. Regulation ofthese events allows preservation of stem cells throughout life, andexpansion of stem cells as well as production of differentiated progenywhen needed for tissue repair.⁽¹⁾ Various embodiments of the presentinvention illustrate that culture of MSCs on an ECM made bymarrow-derived stromal cells promotes symmetric division to produceidentical daughter cells whereas plastic favors production ofdifferentiated progeny by symmetric or asymmetric cell division.Moreover, the MSCs expanded on the marrow ECM retain the ability to forma complete bone like structure comprising a calcified matrix made byosteoblasts, hematopoietic marrow containing adipocytes, and stromalcells that support hematopoiesis and osteoclastogenesis. In contrast,growth of MSCs on tissue culture plastic results in eventual loss ofself-renewal capacity and multipotentiality, and this is associated withexpression of the osteoblast phenotype. Although cells expanded onplastic did form bone in vivo as previously reported,⁽⁸⁾ they made lessbone and minimal hematopoietic marrow.

Culture of MSCs in the presence of three-dimensional (3D) stromal cellderived ECM allows for attachment, self-renewal, and retention ofmultipotentiality of MSCs, whereas culture of MSCs under two-dimensional(2D) conditions with or without certain ECM proteins like type Icollagen or fibronectin does not.

Loss of stem cell properties, coincident with so-called “spontaneous”differentiation when MSCs are cultured on plastic, may actuallyrepresent the response of MSCs to growth factors produced endogenouslyin these cultures. Indeed, autocrine/paracrine production of BMP2/4mediates the production of osteoblastic cells when MSCs are cultured onplastic.⁽⁹⁾ BMPs bind strongly to collagen as well as smallproteoglycans such as biglycan.⁽¹⁰⁾ Embodiments of the present inventiondemonstrate that the ECM sequestered the BMP2 produced by culturedmarrow cells, and this at least partially explains why MSCs retained anundifferentiated phenotype when cultured on a collagenous ECM. Otherpro-differentiating proteins may also be sequestered by the ECM.

MSCs lose their multipotentiality when cultured on tissue cultureplastic. Previous attempts to overcome this limitation have utilizedculture on fibronectin matrices under low oxygen tension (5%)^((11,12))to mimic the microenvironment of the bone marrow,⁽¹³⁾ or culture at lowseeding density in low serum in the presence of growth factors.⁽¹⁴⁻¹⁶⁾These conditions permitted expansion of murine and human MSCs for asmany as 60 population doublings, but the full differentiation potentialand cellular composition of these preparations remains unclear.

The present invention illustrates that the marrow ECM forms part of theniche that supports MSCs in the bone marrow, and that the ECM regulatesthe balance between replication and differentiation in response toappropriate signals. Consequently, the 3D extracellular matrix culturesystem described herein provides a system for the expansion offunctional MSCs for practical applications. This system is invaluablefor identification of the contribution of specific ECM components inregulating the behavior of MSCs. Finally, this system is also useful foridentifying the effect of aging and/or hormonal changes on the abilityof the marrow ECM to maintain MSC function, and thereby contribute tothe development of pathologies such as osteoporosis.

BRIEF SUMMARY OF THE INVENTION

The present inventors disclose mesenchymal stem cells (MSCs) cultured onan ECM made by marrow stromal cells thereby reconstituting the MSCniche. This ECM specifically promotes self-renewal of MSCs and retentionof their multipotentiality. The present inventors demonstrate that themarrow ECM is useful for the maintenance of sternness, and that the ECMprovides a vehicle for MSC expansion.

Cell-free three-dimensional (3D) matrices were prepared from culturedmurine marrow stromal cells. The self-renewal and multipotentiality ofmurine MSCs maintained on this stromal cell-derived ECM were examined invitro and in vivo and compared to MSCs maintained on plastic,fibronectin, Type I collagen, or skin fibroblast-derived ECM.

Stromal cell-derived ECM may comprise collagen types I, III, and V,syndecan-1, perlecan, fibronectin, laminin, biglycan and decorin, andresembles the marrow ECM. This ECM preparation promotes self-renewal ofMSCs, restrains their “spontaneous” differentiation toward theosteoblast lineage, and preserves their ability to differentiate intoosteoblasts or adipocytes in response to BMP2 or rosiglitazone,respectively. In contrast, two-dimensional (2D) matrices made of Type Icollagen or fibronectin, or skin fibroblast-derived 3D ECM, failed to doso. Moreover, transplantation of MSCs expanded on the stromalcell-derived ECM into immunocompromised mice generated five times morebone and eight times more hematopoietic marrow, as compared to MSCsexpanded on plastic.

Thus, the ECM made by bone marrow stromal cells is useful for themaintenance of MSCs and provides a vehicle for the expansion of thesecells ex vivo.

In one embodiment of the present invention, the inventors disclose acellular composition comprising mammalian mesenchymal stem cellsmaintained in culture in an undifferentiated state. The cellularcomposition of mammalian mesenchymal stem cells comprises mammalianmesenchymal stem cells growing on an extracellular matrix.

In another embodiment of the present invention, the inventors disclose acomposition for maintaining mammalian mesenchymal stem cells in culturein an undifferentiated state comprising an extracellular matrix.

The present inventors also disclose a method of maintaining mammalianmesenchymal stem cells in culture in an undifferentiated statecomprising culturing mammalian mesenchymal stem cells in the presence ofan extracellular matrix.

In a further embodiment of the present invention, the inventors disclosea cell culture apparatus for maintaining mammalian mesenchymal stemcells in an undifferentiated state comprising a substrate and anextracellular matrix.

Another method disclosed by the present inventors is a method ofmaintaining mammalian mesenchymal stem cells in an undifferentiatedstate comprising culturing mammalian mesenchymal stem cells in thepresence of an inhibitor of BMP2 activity.

A further embodiment of the present invention is a bone formingcomposition comprising: (i) a cellular composition comprising mammalianmesenchymal stem cells grown on an extracellular matrix, and (ii) atransplantation vehicle.

The present inventors also disclose a method of generating bone in apatient in need thereof comprising: (i) obtaining mammalian mesenchymalstem cells; (ii) producing a bone forming composition by culturing saidmammalian mesenchymal stem cells in vitro on an extracellular matrix andisolating undifferentiated mammalian mesenchymal stem cells from saidculture; and (iii) administration of said bone forming composition tosaid patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates various embodiments of the present inventionincluding a method of manufacturing a composition for maintainingmammalian mesenchymal stem cells in culture in an undifferentiated stateand an exemplary cell culture apparatus for maintaining mammalianmesenchymal stem cells in culture in an undifferentiated state.

FIG. 2A illustrates SEM images of stromal cell-derived ECM before andafter removing cells. The left panels show the ECM made by culturedmarrow stromal cells before and after marrow stromal cell removal at lowmagnification. The right panels, at high magnification, show that thestructure of the ECM is very similar before and after cell removal.Inset: enlargement of high magnification image after cell removal.

FIG. 2B illustrates immunohistochemical staining before and after marrowstromal cell removal for components of cell-free ECM made by culturedmarrow stromal cells. Original magnification: 200×.

FIG. 3A illustrates SEM images of bone marrow cells cultured on tissueculture plastic (top panels) or stromal cell-derived ECM (lower panels)obtained after 5 days of culture.

FIG. 3B illustrates CFU-F number as determined at indicated seedingdensities.

FIG. 3C illustrates the appearance of CFU-F derived from cells culturedinto a cell-free stromal cell-derived ECM, plastic, or 2D fibronectin orType I collagen matrices.

FIG. 3D illustrates the number of cells per CFU-F colony as determinedat indicated seeding densities. *P<0.05, n=3 vs. plastic or the 2Dmatrices containing fibronectin or Type I collagen at the same density.†P<0.05, n=3 compared to plastic or the 2D matrices containingfibronectin.

FIG. 4A illustrates behavior of bone marrow cells cultured on 2D and 3Dmatrices. Primary murine bone marrow cells were placed at 3×10⁵cells/cm² in 6-well plates and cultured on plastic, plastic coated withType I collagen, marrow stromal cell-derived ECM, or skinfibroblast-derived ECM for 25 days. RNA was extracted from the cellcultures at the indicated time points. Total RNA was obtained atindicated days of culture. *P<0.05, n=3 vs. the plastic at the same timepoint.

FIG. 4B illustrates differentiation of bone marrow cells cultured on 2Dand 3D matrices. The appearance of cells cultured on the various culturesystems was observed by phase contrast microscopy after 20 days ofculture. Original magnification: 200×. The arrow indicates nodules ofcells.

FIG. 4C illustrates the level of transcripts of osteoblastic markers asdetermined by TaqMan PCR at the indicated days of culture. *P<0.05, n=3vs. the plastic or plastic coated with Type I collagen at the same timepoint.

FIG. 4D illustrates the level of transcripts for BMP and Wnt antagonistsas determined by TaqMan PCR at the indicated days of culture. *P<0.05,n=3 vs. plastic, Type I collagen, or the stromal ECM at the same timepoint.

FIG. 5A illustrates production of BMP2 in cultures maintained on 2D and3D matrices. Level of BMP2 transcripts in the experiment was determinedas described in FIG. 3A. *P<0.05, n=3 vs. plastic, Type I collagen atthe same time point.

FIG. 5B illustrates production of BMP2 protein in cell/matrix layer orculture supernatant as measured by ELISA assay. *P<0.05, n=3 vs. theplastic.

FIG. 5C illustrates responsiveness to exogenous BMP2 by measuringalkaline phosphatase activity (left panel) and osteocalcin (rightpanel). *P<0.05 (n=3) vs. vehicle control.

FIG. 6A illustrates CFU-F increased by 9-fold, 27-fold, or 48-fold whencultured on plastic, Type I collagen, or the stromal cell-derived ECM,respectively.

FIG. 6B illustrates CFU-OB increased by 1.6-fold, 4-fold, or 9-fold whencultured on plastic, Type I collagen, or the stromal cell-derived ECM,respectively.

FIG. 6C illustrates CFU-AD changed by 0.7-fold, 2.6-fold, or 4.7-foldwhen cultured on plastic, Type I collagen, or the stromal cell-derivedECM, respectively. *P<0.05 by ANOVA vs. the plastic and Type I collagengel, n=3. †P<0.05, n=3 compared to initial isolated.

FIG. 7A illustrates bone formation in vivo by transplanted murine MSCs.Bone was generated by cells pre-cultured on plastic.

FIG. 7B illustrates bone generated by cells pre-cultured on thecell-free marrow stromal cell-derived ECM.

FIG. 7C illustrates marrow like structure containing hematopoieticelements in bone generated by cells pre-cultured on the marrow stromalcell derived ECM.

FIG. 7D illustrates an area from FIG. 7B enlarged to demonstrate anosteoclast with multiple nuclei. For FIGS. 7A through 7D, the followinglegend applies: B, bone; BM, bone marrow with adipocytes andhematopoietic cells; F, fibrous tissue; HA, HA/TCP; OC, osteoclast.

FIG. 7E illustrates the measurement of bone in ossicles. Data shownrepresent the mean (±sd) of bone area calculated from the 3 individualossicles.

FIG. 7F illustrates the area occupied by hematopoietic marrow asdetermined in sections from ossicles obtained 8 weeks after implantationusing the same sections that were used for determination of bone area inFIG. 7E. *P<0.05 vs. bone marrow generated by cells pre-cultured on theplastic, n=3.

FIG. 8 illustrates ECM made by human marrow stromal cells promotescolony forming unit-osteoblast (CFU-OB) and colony formingunit-fibroblast (CFU-F) formation. CFU-F were visualized by crystalviolet shown in blue (right panel). In addition, cells were cultured inosteogenic induction medium (α-MEM containing 15% FCS, 100 μM A2P, 10 mMβ-glycerophosphate, and 10 nM dexamethasone) for 4 weeks, and thenCFU-OB was determined by Von Kossa staining shown in black (left panel).

FIG. 9 illustrates microscopic appearance of CFU-OB.

FIG. 10 illustrates bone formation in vivo by transplanted human MSCs.Bone was generated by cells pre-cultured on the ECM (left panel). Bonewas generated by cells pre-cultured on tissue culture plastic (rightpanel).

FIGS. 11A and B illustrate quantification of bone in ossicles. Eachossicle was bisected. Then, three 10 μm sections were cut from thecenter part at 100 μm intervals. FIG. 11A shows the measurements of bonearea from 3 individual sections for each sample (S1 or S2). FIG. 11Bshows the mean bone area calculated from 3 individual sections for eachsample (S1 or S2).

FIG. 11C illustrates quantification of bone marrow in ossicles with meanbone marrow (hematopoietic tissue) calculated from 3 individual sectionsfor each sample.

DETAILED DESCRIPTION

In various embodiments, the present invention provides a cellularcomposition of mammalian mesenchymal stem cells maintained in culture inan undifferentiated state. The cellular composition of mammalianmesenchymal stem cells may comprise mammalian mesenchymal stem cellsgrowing on an extracellular matrix. The cellular composition may beessentially free of feeder cells. Additionally, the mammalianmesenchymal stem cells may proliferate on the extracellular matrix.

Mammalian mesenchymal stem cells may be obtained from various sources,including, but not limited to, bone marrow or other mesenchymal stemcell sources. Bone marrow cells may be obtained from various sources,such as, for example, iliac crest, femora, tibiae, spine, rib, or othermedullary spaces. Other mesenchymal stem cell sources include, but arenot limited to, embryonic yolk sac, placenta, umbilical cord, fetal andadolescent skin and blood. Isolating and establishing cultures ofmesenchymal stem cells are generally known to those of skill in therelevant art.

In some embodiments of the present invention, the mammalian mesenchymalstem cells forming the cellular composition may be selected from thegroup consisting of human mesenchymal stem cells and murine mesenchymalstem cells.

When maintained or propagated in culture, mammalian mesenchymal stemcells may divide symmetrically or asymmetrically. In some configurationsof the present invention, the mammalian mesenchymal stem cells arecapable of either symmetrical division or asymmetrical division. Inparticular embodiments, the present invention provides materials andmethods which allow for the mammalian mesenchymal stem cells to dividesymmetrically.

In various embodiments of the present invention, a cellular compositioncomprises mammalian mesenchymal stem cells and an extracellular matrix.In particular embodiments of the invention, the extracellular matrixcomprises a marrow stromal cell derived extracellular matrix. Forpurposes of further illustration of the present invention, the marrowstromal cell derived extracellular matrix may be manufactured byobtaining marrow stromal cells, culturing said marrow stromal cells,lysing the marrow stromal cells and removing the lysed marrow stromalcells by washing. The composition that remains after washing is anextracellular matrix that is essentially free of marrow stromal cellsand essentially free of feeder cells. Marrow stromal cells may beobtained and cultured by common methods that are apparent to one ofskill in the relevant art.

The present invention provides for a composition for maintainingmammalian mesenchymal stem cells in culture in an undifferentiatedstate. Said composition comprises an extracellular matrix and saidcomposition is essentially free of feeder cells. Mammalian mesenchymalstem cells of the present embodiment may be selected from the groupconsisting of human mesenchymal stem cells and murine mesenchymal stemcells. Furthermore, the composition comprising an extracellular matrixmay be a marrow stromal cell derived extracellular matrix. In additionto being a marrow stromal cell derived extracellular matrix, theextracellular matrix may also be a three-dimensional matrix.

In various embodiments of the present invention, an extracellular matrixis disclosed and that extracellular matrix is a marrow stromal cellderived extracellular matrix. In particular embodiments, theextracellular matrix may comprise type I collagen, type III collagen,type V collagen, syndecan-1, fibronectin, decorin, biglycan, perlecan,and laminin. As described herein, such an extracellular matrix may bemanufactured by obtaining marrow stromal cells, culturing said marrowstromal cells, lysing the marrow stromal cells and removing the lysedmarrow stromal cells by washing. The composition that remains afterwashing is an extracellular matrix according to various embodiments ofthe present invention.

A further embodiment of the present invention provides a method ofmaintaining mammalian mesenchymal stem cells in culture in anundifferentiated state comprising culturing mammalian mesenchymal stemcells in the presence of an extracellular matrix. The culture ofmammalian mesenchymal stem cells is essentially free of feeder cells. Aspreviously stated, said mammalian mesenchymal stem cells may be selectedfrom the group consisting of human mesenchymal stem cells and murinemesenchymal stem cells. Furthermore, said extracellular matrix may be amarrow stromal cell derived extracellular matrix, and said extracellularmatrix may be a three-dimensional matrix. In various embodiments of thepresent method, said extracellular matrix may comprise type I collagen,type III collagen, type V collagen, syndecan-1, fibronectin, decorin,biglycan, perlecan, and laminin. The method provides for manufacture ofthe extracellular matrix by obtaining marrow stromal cells, culturingmarrow stromal cells, lysing the marrow stromal cells and removing thelysed marrow stromal cells by washing.

Certain embodiments of the present invention provide a cell cultureapparatus for maintaining mammalian mesenchymal stem cells in anundifferentiated state comprising a substrate and an extracellularmatrix. The substrate of the cell culture apparatus may comprise a cellculture container or a substrate that may be placed within a cellculture container. A cell culture container may be selected from thegroup consisting of a flask, a Petri dish, a vat and a reactor. Othercell culture containers may be useful in the present embodiment.

The cell culture apparatus described above comprises an extracellularmatrix, and said extracellular matrix may comprise a marrow stromal cellderived extracellular matrix. Furthermore, said extracellular matrix maybe a three-dimensional matrix. Such an extracellular matrix may comprisetype I collagen, type III collagen, type V collagen, syndecan-1,fibronectin, decorin, biglycan, perlecan, and laminin. Additionally, theextracellular matrix of the cell culture apparatus may be manufacturedby obtaining marrow stromal cells, culturing marrow stromal cells on asubstrate, lysing the marrow stromal cells and removing the lysed marrowstromal cells by washing. The substrate and extracellular matrix thatremains associated with the substrate subsequent to washing may beuseful as a cell culture apparatus. Said cell culture apparatus may beirradiated or treated with chemical agents and stored for periods oftime, such as, for example, approximately one week, approximately onemonth, approximately two months, approximately three months or evenapproximately four months or more. Chemical agents that may be usefulduring such periods of storage include antibiotics and antifungalagents.

Also disclosed is a method of maintaining mammalian mesenchymal stemcells in undifferentiated state comprising culturing mammalianmesenchymal stem cells in the presence of an inhibitor of bonemorphogenetic protein 2 (BMP2) activity. BMP2 is an osteogenic proteinthat belongs to the TGF-β superfamily of proteins and induces osteoblastdifferentiation. An exemplary inhibitor of BMP2 activity is anextracellular matrix that sequesters BMP2. Such an extracellular matrixmay comprise a marrow stromal cell derived extracellular matrix, andsuch a matrix may be a three-dimensional matrix. Additionally, anextracellular matrix with BMP2 inhibiting activity may comprise type Icollagen, type III collagen, type V collagen, syndecan-1, fibronectin,decorin, biglycan, perlecan, and laminin. For purposes of the presentembodiment, the mammalian mesenchymal stem cells may be selected fromthe group consisting of human mesenchymal stem cells and murinemesenchymal stem cells.

Another embodiment of the present invention provides a bone formingcomposition comprising: (i) a cellular composition comprising mammalianmesenchymal stem cells grown on an extracellular matrix, and (ii) atransplantation vehicle. The cellular composition of the presentembodiment is essentially free of feeder cells. Additionally, themammalian mesenchymal stem cells may be either human mesenchymal stemcells or murine mesenchymal stem cells. The extracellular matrix of thecellular composition may comprise a marrow stromal cell derivedextracellular matrix. Additionally, the extracellular matrix of thecellular composition may be a three-dimensional matrix. By way ofexample, the extracellular matrix of the cellular composition maycomprise type I collagen, type III collagen, type V collagen,syndecan-1, fibronectin, decorin, biglycan, perlecan, and laminin.

In order to manufacture the bone forming composition of the presentembodiment, said extracellular matrix may be manufactured by obtainingmarrow stromal cells, culturing marrow stromal cells on a substrate,lysing the marrow stromal cells and removing the lysed marrow stromalcells by washing. The mammalian mesenchymal stem cells may then becultured on the substrate. The mammalian mesenchymal stem cells may thenbe removed from the substrate by techniques known to those of skill inthe art or as described herein. The mammalian mesenchymal stem cells maythen be combined with a transplantation vehicle, such as, for example, atransplantation vehicle comprising hydroxyapatite or an acceptable saltthereof. The transplantation vehicle may further comprise tricalciumphosphate. In various embodiments of the present invention, thetransplantation vehicle may comprise a ceramic powder.

Such a bone forming composition as previously described may beadministered to a mammal in need of bone formation. Such a mammal mayinclude any mammal that has a bone condition requiring bone formation.Exemplary conditions that may require bone formation include, but arenot limited to, such bone conditions as fracture, delayed unions,non-unions, distraction osteogenesis, osteotomy, osseointegration, andosteoarthritis. Mammals that may be benefited by the present embodimentinclude mammals, such as, for example, humans and mice.

A further embodiment of the present invention provides a method ofgenerating bone in a patient in need thereof comprising: (i) obtainingmammalian mesenchymal stem cells; (ii) producing a bone formingcomposition by culturing said mammalian mesenchymal stem cells in vitroon an extracellular matrix and isolating undifferentiated mammalianmesenchymal stem cells from said culture; and (iii) administration ofsaid bone forming composition to said patient. The mammalian mesenchymalstem cells of the present embodiment may be selected from the groupconsisting of human mesenchymal stem cells and murine mesenchymal stemcells. Additionally, the extracellular matrix of the present embodimentmay comprise a marrow stromal cell derived extracellular matrix aspreviously described. In some configurations, the mammalian mesenchymalstem cells of the present embodiment may be allogeneic or autologous tosaid patient. Moreover, the patient may be a human patient, and inparticular embodiments, the patient may be an aging human patient. Anaging patient may be, for example, any patient who is at least 35 yearsold, at least 40 years older, at least 50 years old, or at least 60years old, or older. In various aspects of the present embodiment, thebone forming composition may further comprise a transplantation vehicle,such as, for example, a ceramic powder. Such ceramic powders that areuseful in the present invention include hydroxyapatite ceramic powders.Furthermore, the transplantation vehicle may comprise tricalciumphosphate.

The bone forming composition may be administered to a patient in needthereof concurrently with surgery for a bone condition,laparoscopically, by injection or by any other means known to one ofskill in the relevant art. The bone forming composition may beadministered locally or systemically.

In this manner, mesenchymal stem cells may be expanded and propagated invitro and subsequently administered to a patient thereby providing thepatient with a sufficient quantity and quality of mesenchymal stem cellssuch that the patient may be effectively treated for a conditionrequiring bone formation, bone remodeling, bone development or any othercondition that is improved by the action of mesenchymal stem cells.

EXAMPLES

The following examples are further illustrative of the presentinvention, but it is understood that the invention is not limitedthereto.

Animals. Swiss Webster female mice, 6-8 weeks old, were obtained fromHarlan (Indianapolis, Ind.). The University of Arkansas for MedicalSciences Division of Laboratory Animal Medicine approved the animal useprotocol.

Scanning electron microscopy. Samples were washed three times with PBSand fixed with 2% glutaraldehyde in 0.1M sodium cacodylate buffer (pH7.2) for one hour and then transferred to 0.1 M cacodylate buffersolution. The specimens were dehydrated in ascending concentrations ofethanol (from 70% to 100%), embedded in peon resin (Poly/bed 812Polysciences Int., Warrington, Pa.), and then coated with gold andpalladium. After dehydration the coverslips were attached to a stub andsputtered with gold-palladium. The gold-palladium-coated cultures wereexamined using an FEI/Philips XL30 Field emission environmental scanningelectron microscope (Hillsboro, Oreg.).

Immunohistochemistry. Stromal cell-derived ECM, before or after removalof cells, was fixed for 30 minutes with 4% formaldehyde in PBS at roomtemperature, washed with PBS, and blocked with 5% normal goat serumcontaining 0.1% BSA in PBS for one hour. The matrices were thenincubated with the relevant primary antibodies (1:10 dilution) in 2%goat serum for two hours. Antibodies against biglycan, collagen type I,III, V, fibronectin, decorin, perlecan, syndecan-1, and laminin, werepurchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).Non-specific isotype IgG (1:10 dilution) was used as a negative control.After washing with PBS, samples were incubated with the appropriatehorseradish peroxidase-conjugated secondary antibody (1:100 dilution)for one hour, developed with a 3,3′-diaminobenzidine substrate-chromogensystem (Dako Corp., Carpinteria, Calif.) for five minutes, and thencounterstained with methyl green.

Determination of colony forming unit fibroblast (CFU-F), osteoblast(CFU-OB), and adipocyte (CFU-AD). Freshly isolated murine femoral marrowcells were plated into 6-well plates at the indicated seeding densities,incubated for four hours at 37° C. to allow attachment of adherentcells, and washed twice with PBS to remove the non-adherent cells. Then,irradiated guinea pig feeder cells (3×10⁶) were added immediately in 4ml of the α-MEM medium described above containing 1 mML-ascorbate-2-phosphate (Wako Chemicals, Richmond, Va.). Afterapproximately 10 to 12 days (CFU-F) or 25 days (CFU-OB), colonies werevisualized with crystal violet or Von Kossa staining, respectively. Fordetermination of CFU-AD, 100 nM rosiglitazone or vehicle(dimethylsulfoxide) was added to the cell cultures at day seven. On day25, the cultures were stained with Von Kossa to visualize coloniescontaining mineralizing osteoblasts and with Oil Red O to visualizeadipocytes. Colonies containing more than 50 cells were counted using adissecting microscope.

Measurement of MSC self-renewal has been previously described.⁽¹⁷⁾Briefly, freshly isolated bone marrow cells were pre-cultured onto6-well plates with or without the cell-free ECM or pre-cultured in atype I collagen gel at 7×10⁶ cells per well for 7 days. Cells werecollected following treatment with collagenase and reseeded ontostandard tissue culture plastic with irradiated guinea pig feeder cellsin 4 ml of the α-MEM medium described above containing 1 mML-ascorbate-2-phosphate for CFU-F, CFU-OB, and CFU-AD assays.

Quantification of gene expression in cultured bone marrow cells. TotalRNA was extracted using Ultraspec reagent (Biotecx Laboratories, Inc.,Houston, Tex.). RNA (2 μg) was reverse-transcribed using a High CapacitycDNA Archive Kit (Applied Biosystems, Foster City, Calif.). Thetranscripts of interest, and that of the housekeeping gene GAPDH, wereamplified from cDNA by real-time PCR using TaqMan Universal PCR MasterMix and Assay Demand or Assay by Design primer and probe sets (AppliedBiosystems). Amplification and detection were carried out with an ABIPrism 7300 Sequence Detection System (Applied Biosystems) as follows:denaturation at 95° C. for 10 minutes, 40 cycles of amplificationincluding denaturation at 94° C. for 15 seconds and annealing/extensionat 60° C. for one minute. Gene expression was quantified by subtractingthe GAPDH threshold cycle (Ct) value from the Ct value of the gene ofinterest, and expressed as 2^(−ΔCt), as described by the protocol of themanufacturer.

Measurement of alkaline phosphatase (ALP) activity and osteocalcinsecretion in response to BMP2. Freshly isolated murine bone marrow cellswere cultured in α-MEM described above for 15 days. For measurement ofALP response, FBS was reduced to 2% and then human recombinant BMP2 (R&DSystems, Inc., Minneapolis, Minn.) was added. After 48 hours, cells werelysed (20 mM Tris, 0.5 mM MgCl₂, 0.1 mM ZnCl₂ and 0.1% Triton X) and ALPactivity was determined using an alkaline phosphatase kit (SigmaChemical Co., St. Louis, Mo.). The ALP value was normalized by theamount of protein in the lysates, and was expressed as ALPactivity/minute/μg. For measurement of the osteocalcin response, mediumwas removed six days after addition of BMP2, and the osteocalcin levelswere measured by RIA (Biomedical Technologies Inc., Stoughton, Mass.).

Measurement of BMP2. Murine bone marrow cell cultures were establishedon plastic or on the marrow stromal cell-derived ECM in 6-well plates.After 15 days, the supernatant was collected. After extensive rinsing,BMP2 was extracted from the ECM/cell layer using 2M urea, 2% SDS, 10%glycerol and 10 mM Tris-HCl pH 6.8.⁽¹⁸⁾ The amount of BMP2 in theculture supernatant and the extracts was measured using a murinespecific ELISA Assay Kit (R&D Systems, Minneapolis, Minn.).

In vivo bone formation. Marrow cells were cultured for seven days onplastic or the stromal cell-derived ECM. Adherent cells (1×10⁶) wereloaded into a transplantation vehicle such as, for example,hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Inc,Warsaw, Ind., USA), and transplanted subcutaneously into the dorsalsurface of 10-week-old immunodeficient beige mice (NIH-bg-nu-xid, HarlanSprague Dawley, Indianapolis, Ind.), as previously described.^((8,19))Three transplants were made for each pre-culture system. Transplantswere harvested after four or eight weeks, fixed in 10% phosphatebuffered formalin at 4° C. for 24 hrs, decalcified with 5% EDTA (pH8.0)at room temperature for 1-2 weeks, and then embedded in paraffin. Eachossicle was bisected, and three sections (10 μm) were cut from each partat 100 μm intervals. A total of nine H&E stained sections were used forquantification. The percentage of the area of new bone and hematopoieticmarrow formed in transplants was measured by using Osteometrics imageanalysis software (Ostomeasure version 3.00, Osteometrics Inc., Atlanta,Ga.).

Statistical analysis. All data are presented as mean±standard deviation.Statistical analyses were done by using Student's t test or one-wayANOVA. Differences of P<0.05 were considered significant.

Example 1

FIG. 1 illustrates an exemplary method for manufacturing a cell cultureapparatus for maintaining or propagating MSCs in culture in anundifferentiated state.

Freshly isolated murine femoral marrow cells were seeded onto tissueculture plastic at 3×10⁵ cells/cm², and cultured for seven days in α-MEM(Life Technologies, Grand Island, N.Y.), supplemented with glutamine (2mM), penicillin (100 U/ml), streptomycin (100 μg/ml) (Sigma ChemicalCompany, St. Louis, Mo.), and 15% pre-selected fetal bovine serum (FBS,Atlanta Biologicals, Lawrenceville, Ga.). For preparation of skinfibroblasts, the ventral skin from 2-5 day old mice were removed, rinsedin PBS, and cut into 1-mm² pieces. The tissue was incubated with 400U/ml collagenase for 40 minutes at 37° C., rinsed with PBS, and culturedin high glucose DMEM medium containing 10% FBS, glutamine (2 mM) andpenicillin (100 U/ml) until primary fibroblasts migrated out of thesamples onto the culture plates reaching 70% confluence. Fibroblastswere collected, and frozen for storage or used between passages two andsix for the establishment of ECM.

To prepare ECM, cells were seeded onto Thermanox plastic cover slipscoated with fibronectin at 1×10⁴ cells/cm², and cultured for seven daysin the α-MEM medium described above. Then ascorbic acid (50 μM) (SigmaChemical Company, St. Louis, MO) was added to the cell cultures for anadditional eight days. After extensive washing with PBS, cells wereremoved from the ECM by incubation with 0.5% Triton X-100 containing 20mM NH₄OH in PBS for five minutes at 37° C. The ECM was then treated withDNase at 100 μ/ml (Sigma Chemical Company, St. Louis, MO) for one hourat 37° C. The plates were washed with PBS three times, then 2.0 ml ofPBS containing 50 μg/ml gentamicin and 0.25 μg/ml Fungizone was added tothe plates. and the plates were stored at 4° C. up to four months.

Example 2

Preparation of a marrow stromal cell-derived ECM. Scanning electronmicroscopy (SEM) revealed that stromal cells cultured from murinefemoral bone marrow elaborated a fibrillar ECM (FIG. 2A). Prior tostudying the behavior of MSCs on this ECM, the stromal cells were lysedwith 0.5% Triton X-100 containing 20 mM NH₄OH followed by DNasetreatment to digest remaining nuclear contaminants.⁽²⁰⁾ The resulting 3Dmatrix contained fibers of approximately 25 nm diameter and wasapproximately 100 μm thick as determined by transmission electronmicroscopy (data not shown).

When examined prior to removal of stromal cells, immunostaining revealedhigh levels of collagen types I, III, V, syndecan-1, perlecan,fibronectin, laminin, biglycan and decorin associated with both stromalcells and the ECM (FIG. 2B). The protein composition of the ECM was onlymodestly affected by the cell extraction procedure as indicated byretention of immunostaining for all of the proteins that were examinedexcept for collagen type V (FIG. 2B).

Example 3

Culture on stromal cell-derived ECM facilitates retention of MSCproperties. The ECM affects MSC adherence and proliferation. MSCs weredetected and quantified by their ability to form a colony offibroblastic cells.⁽²¹⁾ These colony-forming cells, called colonyforming unit-fibroblasts (CFU-F), comprise MSCs. After five days ofculture, most of the cells in the colony were embedded inside of thecollagenous matrix and exhibited a fibroblastic morphology withextensive cellular processes. In contrast, cells cultured on tissueculture plastic were round and flat (FIG. 3A).

When cultured on the stromal cell-derived ECM, there was approximately atwo to three fold increase in the number of CFU-F as compared to tissueculture plastic, demonstrating that the ECM promoted MSC attachment(FIGS. 3B and 3C). 2D ECM preparations, made by coating tissue cultureplasticware with fibronectin or Type I collagen, were less effective(FIGS. 3B and 3C). Moreover, the colonies that developed on the stromalcell-derived ECM contained approximately four-fold more cells thancolonies that developed on plastic or fibronectin, whereas coloniesformed on Type I collagen matrix contained only approximately two-foldmore cells than the colonies that developed on plastic or fibronectin(FIG. 3D). These findings indicate that a collagen-containing ECMuniquely promotes the proliferative capacity of MSCs and/or theirtransit amplifying progeny.

Cells in parallel cultures were detached by treating with 400 U/mlcollagenase and the total number of cells per well was counted using ahemocytometer. The mean number of cells per colony was estimated bydividing the number of cells per well by number of colonies per well.

Example 4

The present inventors further demonstrate that the marrow stromalcell-derived ECM prevented “spontaneous” differentiation of MSCs. The 2DType I collagen ECM, and a 3D skin fibroblast-derived ECM (SF-ECM)elaborated by skin fibroblasts obtained from neonatal mice were used ascontrols. The latter ECM exhibited a fibrillar structure similar to thatof marrow stromal cell-derived ECM (data not shown), consistent with thepresence of type I and type III collagens. The proliferation of marrowcells placed on these matrices was similar, as determined by RNAcontent, and was increased as compared to cells cultured on plastic(FIG. 4A). When cultured on plastic for 20 days, cells were grouped intonodules whereas cells cultured on the collagen-containing ECMpreparations were evenly distributed and exhibited a uniform morphology(FIG. 4B). The expression of the osteoblast markers alkalinephosphatase, collal, bone sialoprotein, and osteocalcin progressivelyincreased during 25 days of culture (FIG. 4C), consistent with the“spontaneous” differentiation of MSCs reported previously.⁽²²⁾ Incontrast, stromal cell-derived or skin fibroblast-derived ECMpreparations prevented or delayed the appearance of these osteoblastmarkers. The 2D Type I collagen ECM also retarded osteoblastogenesis,but it was less effective. In a separate experiment, there was minimalmineral deposition, as determined by Von Kossa staining, when cells weremaintained on the stromal cell-derived ECM (data not shown).

The restraint of osteoblastogenesis seen in cultures of MSCs maintainedon stromal cell-derived ECM did not appear to be due to increasedproduction of antagonists of the bone morphogenetic proteins (BMPs) andWnt proteins needed for osteoblast differentiation. Specifically, thelevel of Sost, Noggin, Dkk1, Chordin, Gremlin, and Twisted gastrulationtranscripts in cultures maintained on this ECM were equivalent to, orless than, that of cells cultured on plastic (FIG. 4D). A similarpattern was seen in the case of cells cultured on Type I collagen. Onthe other hand, transcripts of most of these antagonists were higher incells cultured on the skin fibroblast-derived ECM, except for Gremlin 2(FIG. 4D).

The marrow stromal cell-derived ECM supported MSC function, whereas theECM made by skin fibroblasts failed to support responsiveness toexogenous BMP2. The transcript levels of BMP and Wnt antagonists wereincreased in these cultures.

Example 5

Autocrine/paracrine production of BMP2 mediates the osteoblastogenesisthat occurs when MSCs are cultured on plastic in the presence of highascorbic acid.⁽⁹⁾ Hence, the restraint of osteoblast differentiationobserved in cultures maintained on the stromal cell-derived ECM couldhave been due to decreased synthesis of endogenous BMP2. The level ofBMP2 transcripts, however, was similar to or higher in culturesmaintained on the stromal cell-derived or skin fibroblast-derived ECM ascompared to cells maintained on plastic (FIG. 5A), making thispossibility unlikely. Murine bone marrow cell cultures were establishedon plastic or on the stromal cell-derived ECM in 6-well plates. After 15days, the supernatant was collected. BMP2 was extracted from theECM/cell layer using 2M urea, 2% SDS, 10% glycerol and 10 mM Tris-HCl pH6.8. BMP2 in the supernatant and in the ECM/cell layer extract wasquantified by ELISA.

The cells maintained on the 2D type I collagen ECM expressed low levelsof BMP2 compared to the other cultures. A separate experimentdemonstrated that the amount of BMP2 protein was increased approximately2-fold in cultures maintained for 15 days on the stromal cell-derivedECM as compared to plastic (FIG. 5B), and that >90% of BMP2 protein wasassociated with the cell/matrix in cultures maintained on the stromalcell-derived ECM as compared to only 60% in the case of culturesmaintained on the plastic. Thus, the restraint of osteoblastdifferentiation when MSCs were cultured on this ECM is related tosequestration of BMP2 by the ECM. Moreover, the expression of BMP2R1Btranscripts was increased when cells were cultured on collagenous ECM ascompared to plastic, indicating that lack of BMP2 receptor does notaccount for the poor responsiveness of cultures maintained on Type Icollagen or skin fibroblast-derived ECM (data not shown).

Although MSCs did not undergo “spontaneous” osteoblastogenesis whencultured on the stromal cell-derived ECM, they were capable ofdifferentiating into osteoblasts in response to exogenous BMP2. Whenadded 15 days after establishment of the cultures, as little as 3 ng/mlor as little as 10 ng/ml of BMP2 stimulated alkaline phosphataseactivity and osteocalcin secretion (FIG. 5C). Consistent with the dataof FIG. 4C, which shows an increase in alkaline phosphatase transcripts,basal alkaline phosphatase activity was elevated in cultures maintainedon tissue culture plastic as compared to the ECM. Addition of exogenousBMP2 to cells maintained on plastic modestly increased alkalinephosphatase activity, as well as osteocalcin secretion, but theseeffects required 10-fold higher concentrations than the cells culturedon the ECM. BMP2 increased alkaline phosphatase activity, but notosteocalcin secretion, in MSCs maintained on the 2D Type I collagen ECM.MSCs failed to respond to exogenous BMP2 when cultured on skinfibroblast-derived ECM.

Murine bone marrow cell cultures were established either on plastic orplastic coated with a collagenous matrix including marrow stromalcell-derived ECM, skin fibroblast-derived ECM or Type I collagen. After15 days of culture, human recombinant BMP2 was added at the indicatedconcentrations. Alkaline phosphatase activity was determined after twodays. Osteocalcin from conditioned medium was measured by RIA after sixdays.

Example 6

Culture of MSCs on stromal cell-derived ECM promotes self-renewal andretention of multipotentiality. The self-renewal of MSCs was determinedusing a re-plating assay in which the increase in colony forming cellsfollowing seven days of pre-culture of MSCs was quantified.⁽¹⁷⁾Self-renewal of MSCs was measured for MSCs cultured on plastic, the 3Dstromal cell-derived ECM, or 3D Type I collagen gels that have beenpreviously described.⁽⁹⁾ ECM from skin fibroblasts was not examined asBMP2 responsiveness of MSCs was lost in such cultures. The number ofCFU-F colonies was increased approximately 48-fold when the cells werepre-cultured on stromal cell-derived ECM as compared approximately9-fold or approximately 27-fold in cultures maintained on plastic orType I collagen gel, respectively (FIG. 6A). Self-renewal of MSCs.Murine bone marrow cells were cultured on plastic, or 3D Type I collagengel, or the stromal cell-derived ECM at 5×10⁶ cells per 10 cm² well.Some of the bone marrow cells were used to determine the number ofCFU-F, CFU-OB, and CFU-AD present in the initial isolate. After sevendays of pre-culture, the adherent cells were detached and harvested withcollagenase, and reseeded into tissue culture plastic for measuringCFU-F, CFU-OB and CFU-AD.

Similarly, the replication of colony-forming progenitors capable ofdifferentiating into osteoblasts [CFU-osteoblast (CFU-OB)] and/oradipocytes [CFU-adipocyte (CFU-AD)], was significantly higher when. MSCswere pre-cultured on the stromal cell-derived ECM, as compared to cellscultured on plastic or Type I collagen gel. Indeed, CFU-OB did notsignificantly increase when pre-cultured on plastic, consistent with theevidence of FIG. 4C that MSCs divided and differentiated toward theosteoblast lineage, instead of dividing to produce identicalcolony-forming MSCs.

The proportion of CFU-OB and CFU-AD among the entire population ofcolony-forming MSCs (as detected by CFU-F) declined approximately 3-foldduring expansion, from approximately 50% in the initial marrow cellisolate to approximately 15% after pre-culture on plastic, Type Icollagen gel, or stromal cell-derived ECM (FIGS. 6B and 6C). This mayreflect the heterogeneity of the colony forming cells present in theinitial isolate, and the fact that some of the progenitors in the CFU-Fpopulation divided more frequently than others during the pre-cultureperiod.

Example 7

In view of the likely heterogeneity of the colony forming cellpopulation, the inventors compared the capacity of MSCs expanded onplastic or the stromal cell-derived ECM to form bone and hematopoieticmarrow in vivo using a transplantation assay.⁽¹⁹⁾ Following seven daysof culture on plastic or on stromal cell-derived ECM, the cells wereloaded onto a hydroxyapatite/tricalcium phosphate (HA/TCP) carrier andimplanted subcutaneously into immuno-compromised NIH-bg-nu-xid mice. Theamount of bone generated at eight weeks after implantation by MSCspre-cultured on plastic was approximately 3% of bone of the total areaof the ossicle. However, there was minimal hematopoietic marrow, andadipocytes and osteoclasts were rarely observed (FIGS. 7A, 7B and 7E).Importantly, MSCs pre-cultured on stromal cell-derived ECM generatedapproximately five times more bone than the cells pre-cultured on tissueculture plastic (FIGS. 7B through 7E), which corresponds with theapproximately 5-fold greater increase in CFU-OB replication duringpre-culture on the ECM as compared to plastic (FIG. 6B).

Bone marrow cells were pre-cultured for seven days on plastic or thestromal cell-derived ECM. The cells were then loaded onto HA/TCP andimplanted subcutaneously into the dorsal surface of 10-week-oldimmunodeficient beige NIH-bg-nu-xid mice. Three transplants were madefor each group. The transplants were harvested after four or eightweeks, fixed, decalcified and then processed for paraffin embedding.

Osteoclasts were also present in ossicles made by cells pre-cultured onthe ECM (FIG. 7D), indicating the presence of stromal cells that supportosteoclast differentiation. Extensive hematopoietic marrow characterizedby a large number of adipocytes was observed at 8, but not 4, weeksafter implantation (FIG. 7C). The area of hematopoietic marrow wasincreased by 8-fold in ossicles made by cells cultured on the ECM ascompared to cells cultured on plastic (FIG. 7F). Each ossicle wasbisected. Then, 10 μm sections were cut from the bisection point of oneportion at 100 μm intervals for measurement of the mean bone area foreach ossicle.

Example 8

Primary human bone marrow mononuclear cells (hBMCs, purchased fromAllCells, LLC.) were placed onto either the ECM made by human marrowstromal cells (hMSC-ECM) or tissue culture plastic at various cellseeding densities (2, 1, and 0.5×10⁶ cells per well). After 4 hours ofincubation, the non-adherent cells were removed by rinsing with PBSonce. Then the cells were cultured in α-MEM containing 15% FCS for 2weeks.

FIG. 8 illustrates ECM made by human marrow stromal cells promotescolony forming unit-osteoblast (CFU-OB) and colony formingunit-fibroblast (CFU-F) formation. CFU-F were visualized by crystalviolet shown in blue (right panel). In addition, cells were cultured inosteogenic induction medium (α-MEM containing 15% FCS, 100 μM A2P, 10 mMβ-glycerophosphate, and 10 nM dexamethasone) for 4 weeks, and thenCFU-OB was determined by Von Kossa staining shown in black (left panel).

Example 9

The colonies formed by cells cultured on the ECM contained bothosteoblasts as visualized by the deposition of mineral stained with VonKossa (black), and adipocytes stained with Oil Red O (red). The coloniesformed by cells cultured on tissue plastic contained less mineralcontent and fewer adipocytes. FIG. 9 illustrates microscopic appearanceof CFU-OB.

Example 10

Primary human bone marrow mononuclear cells (AllCells, LLC.) werepre-cultured for 14 days on tissue culture plastic or the human stromalcell-derived ECM. The cells were then loaded onto a transplantationvehicle [hydroxyapatite/tricalcium phosphate (HA/TCP) particles] andimplanted subcutaneously into the dorsal surface of 10 weeks oldimmunodeficient beige NIH-bg-nu-xid mice. The transplants were harvestedafter 8 weeks, fixed, decalcified and then processed for paraffinembedding.

FIG. 10 illustrates bone formation in vivo by transplanted human MSCs.Bone was generated by cells pre-cultured on the ECM (left panel). Bonewas generated by cells pre-cultured on tissue culture plastic (rightpanel).

Example 11

FIGS. 11A and B illustrate quantification of bone in ossicles. Eachossicle was bisected. Then, three 10 μm sections were cut from thecenter part at 100 μm intervals. FIG. 11A shows the measurements of bonearea from 3 individual sections for each sample (S1 or S2). FIG. 11Bshows the mean bone area calculated from 3 individual sections for eachsample (S1 or S2).

FIG. 11C illustrates quantification of bone marrow in ossicles with meanbone marrow (hematopoietic tissue) calculated from 3 individual sectionsfor each sample.

REFERENCES

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All references cited in this specification are hereby incorporated byreference in their entirety. The discussion of the references herein isintended merely to summarize the assertions made by their authors and noadmission is made that any reference constitutes prior art relevant topatentability. Applicant reserves the right to challenge the accuracyand pertinence of the cited references.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description be interpreted asillustrative and not in a limiting sense. Unless explicitly stated torecite activities that have been done (i.e., using the past tense),illustrations and examples are not intended to be a representation thatgiven embodiments of this invention have, or have not, been performed.

What is claimed is:
 1. A bone forming composition comprising: (i) acellular composition comprising undifferentiated mammalian mesenchymalstem cells that have been cultured on and then removed from athree-dimensional marrow stromal cell derived extracellular matrix, and(ii) a transplantation vehicle; wherein said extracellular matrixcomprises type I collagen, type II collagen, type V collagen,syndecan-1, fibronectin, decorin, biglycan, perlecan, and laminin; andwherein said transplantation vehicle comprises a ceramic powder.
 2. Thebone forming composition of claim 1 wherein said mammalian mesenchymalstem cells are selected from the group consisting of human mesenchymalstem cells and murine mesenchymal stem cells.
 3. The bone formingcomposition of claim 1 wherein said extracellular matrix is manufacturedby culturing marrow stromal cells, lysing the marrow stromal cells andremoving the lysed marrow stromal cells by washing.
 4. The bone formingcomposition of claim 1 wherein said transplantation vehicle comprises ahydroxyapatite ceramic powder.
 5. The bone forming composition of claim1 wherein said transplantation vehicle further comprises tricalciumphosphate.
 6. A method of generating bone in a patient in need thereofcomprising: (i) obtaining mammalian mesenchymal stem cells; (ii)producing a bone forming composition by culturing said mammalianmesenchymal stem cells in vitro on a three-dimensional marrow stromalcell derived extracellular matrix, isolating undifferentiated mammalianmesenchymal stem cells from said culture, and combining saidundifferentiated mammalian mesenchymal stem cells with a transplantationvehicle; and (iii) administration of said bone forming composition tosaid patient; wherein said extracellular matrix comprises type Icollagen, type II collagen, type V collagen, syndecan-1, fibronectin,decorin, biglycan, perlecan, and laminin; and wherein saidtransplantation vehicle comprises a ceramic powder.
 7. The method ofclaim 6 wherein said mammalian mesenchymal stem cells are selected fromthe group consisting of human mesenchymal stem cells and murinemesenchymal stem cells.
 8. The method of claim 6 wherein saidextracellular matrix is manufactured by culturing marrow stromal cellson a substrate, lysing the marrow stromal cells and removing the lysedmarrow stromal cells by washing.
 9. The method of claim 6 wherein saidmammalian mesenchymal stem cells are autologous to said patient.
 10. Themethod of claim 6 wherein said patient is a human patient.
 11. Themethod of claim 10 wherein said human patient is an aging human patient.12. The method of claim 6 wherein said transplantation vehicle comprisesa hydroxyapatite ceramic powder.
 13. The method of claim 6 wherein saidtransplantation vehicle further comprises tricalcium phosphate.
 14. Themethod of claim 6 wherein said administration is selected from the groupconsisting of local administration or systemic administration.